Our objectives are to understand, at the molecular level, the mechanisms which are responsible for temporal expression and cell differentiation in Bacillus subtilis. Gene fragments which express vegetative and sporulation functions, will be isolated by making use of the recently developed technology of restriction enzymes and plasmids. Preliminary experiments will be carried out using recombinant molecules of an E. coli plasmid. Hybrid clones will be screened within a low-risk biohazard bacility to determine the genetic content of B. subtilis DNA carried by the molecular vehicle. A substantial effort is being made to develop a similar system in B. subtilis. This will provide us with a homologous genetic system and allow us to do direct screening for sporulation markers as well as a complementation analysis of subtilis genetic markers. These experiments would be impossible using the known system in E. coli. Purified gene fragments of known genetic content, obtained either from an E. coli plasmid, or a subsequently developed B. subtilis vehicle, will be analyzed by RNA hybridization of mRNA made during vegetative growth and during sporulation.